White and other fur colourations and hybridization in golden jackals (Canis aureus) in the Carpathian basin

The golden jackal (Canis aureus) is a reoccurring species in the centre of the Carpathian basin, in Hungary. In total, 31 golden jackal tissue samples were collected, from 8 white-coated, 2 black-coated and one mottled animal across Hungary. Sequences and fragment length polymorphisms were studied for white colour (MC1R), and for black coat colouration (CBD103). In each white animal, the most widespread mutation causing white fur colour in dogs in homozygous form was detected. Three animals were found to carry the mutation in heterozygous form. The two black golden jackals were heterozygous for the 3 bp deletion in CBD103 that mutation for black coat colouration in dogs, and one of them also carried the mutation causing white fur. None of the white animals showed signs of hybridization, but both the black and the mottled coloured individuals were found to be hybrids based on genetic testing. Kinship was found three times, twice between white animals, and once between a white animal and an agouti animal carrying the mutation of white coat. Our results confirm the findings that golden jackal–dog hybrids may occur without human intervention, and the detected mutation causing white fur colour in golden jackals could possibly be due to an early hybridization event.

genomic differences 16,17 .Even though most wolf species fall under the Endangered Species Act of the USA 18 , there have been several attempts to establish wolf-dog hybrid breeds, such as the Calupoh 19 .
Coyotes are known to interbreed with dogs 20,21 , and genetic analyses of mitochondrial DNA show that dog ancestry is widespread in coyotes in the southeastern United States 14 .In 2010, Kays et al. 22 found that eastern coyotes interbreed with wolves in the northeastern area of the US.They also found that this hybridization contributed to a larger body size, which resulted in better hunting abilities regarding deer, and the larger coyotes filled a vacant niche in the area left by the eradication of wolves 15,23 .These coyote-wolf hybrids are generally known as coywolves 24 , but whether they should be considered a subspecies is still heavily debated 22 .
Hybridization with dogs has caused several phenotypic changes in wolves, such as black coat colouration in North American grey wolves, which was introduced to the species at least 7500 years ago through introgression with dogs 25 , but there have also been documented cases of hybridization between dogs and coyotes 25 and wolves in North America 26 resulting in several morphological changes.
There is no direct evidence of hybridization between grey wolves (Canis lupus) and golden jackals in the wild, but there is genetic evidence of admixture between golden jackals and African wolves (Canis lupaster) in Israel 27 .
Historical anecdotes 28,29 can be found about possible jackal-dog hybrids.Erik Ziman described breeding experiments with poodles and jackals 30 , and well-documented evidence for human-made dog-jackal hybrids are the Sulimov dogs 31,32 .Recently in India, camera trap pictures have shown golden jackals showing dog-like pelt colouration in urban areas 33 , which could be due to possible hybridization.One documented instance of hybridization in the wild was confirmed by genetic studies, which was described by Galov et al. in 34 .
Phenotypic changes resulting in pheomelanistic coat due to MC1R mutations are observed in many mammal species [35][36][37] , but a loss-of-function mutation is also responsible for the white or golden colouration of dogs' fur colour 38 .The same mutation could also be found in coyotes 39 in North America.
In dogs, there are two widespread MC1R mutations: the first is the dominant E m (melanistic mask), which is caused by a valine to methionine change at amino acid 264, resulting in a melanistic mask on the face, dark underside and legs 40 .The other is a "recessive yellow" allele "e", which changes an arginine to a premature stop codon at amino acid position 306, and is considered recessive 38 , therefore can remain hidden for generations without selection 41 .Breed-specific mutations exist in sighthounds 42 , English Cocker Spaniels, huskies and Australian cattle dogs 43 , but they have not been found in wild canids yet.
Black or melanistic coat colour in dogs is caused by a 3-basepair in-frame deletion (ΔG23) in the canine beta-defensine 103 gene (CBD103) 44,45 .This ΔG23 has been found in North American wolves 25 , Italian wolves 46 , coyotes 47 , and in golden jackal-dog hybrids described by Galov et al. 34 .Melanism is considered a dominant trait, therefore one copy of the mutation is enough to create a phenotypic change with a properly working melanocortin-1 receptor.
Merle coat colouration in dogs is caused by the insertion of a short interspersed element (SINE) into the SILV gene and results in diluted areas of eumelanistic fur, showing a mottled phenotype in heterozygous form 48,49 , whereas in homozygous form it associates with ocular abnormalities and deafness 50 .Clark et al. reported that several dogs with non-merle phenotype were heterozygous for a smaller insertion, and Langevin et al. found that length differences in the poly-A tail of the SINE insertion in the SILV gene results in differences in the merle phenotypes of dogs that can be categorized into six groups based on the poly-A tail length (Mc, Mc +, Ma, Ma +, M, Mh) 51 .
Recently there have been several documented and legally shot white-coloured golden jackals in the southern areas of Hungary, as well as black and mottled animals.Our aim in this study was to determine the mutations behind their fur colour, and to detect whether the change in their phenotype was due to possible hybridization with dogs, using microsatellite markers and mitochondrial sequencing.
Four SNPs were present in our samples, c.475C>T, c.476C>A, c.790A>G and c.916C>T; the latter of these is the stop codon mutation for white coat colouration in dogs.
At c.475, where most agouti-coloured golden jackals are homozygous for the T allele, which results in a proline to serine amino acid change, one animal showed heterozygosity.At c.476, white-coloured jackals are all homozygous for the A allele, resulting in a proline to glutamine amino acid change.We found two instances of heterozygosity of c.790A>G, which is the mutation causing masked phenotype in dogs.At c.916, all white animals (Fig. 1) were homozygous for the T allele, which results in a stop codon terminating the open reading frame of the melanocortin receptor gene.We also found three animals heterozygous for the c.916C>T mutation.
Analysing the other previously described SNPs in the MC1R sequences 38 , at Ser90Gly (c.268) all of our samples were homozygous for the A allele, at Ala105Thr (c.313) they were all homozygous for the G allele, and at Arg301Cys (c.901) all samples were homozygous for the C allele (Supplementary Table 1).

CBD103 results
Not only black, but also white and mottled jackals were genotyped for the ΔG23 mutation of the CBD103 gene, since the 916C>T MC1R mutation is epistatic in homozygous form to this mutation.Using fragment analysis, we found that the black animals (Fig. 2) were heterozygous for the 3-basepair deletion, and all other animals were homozygous for the wild-type allele (Supplementary Table 1).

SILV results
In the mottled animal's case (Fig. 3), the phenotype was highly resemblant to agouti or dark sable merle dogs' look that is caused by a SINE insertion in the SILV gene.Using fragment analysis, we found a peak at 434 basepairs with a wild-type allele of 171 basepairs, which means the animal has a 263-basepair SINE insertion, which categorizes it as Ma +, atypical merle animal.

Partial mitochondrial DNA sequencing
We analysed the mitochondrial D-loop sequence of the 31 animals acquired for this research using BLAST.In 30 cases the results showed golden jackal origins; sample 5928HybB, however, was identified as a dog (GenBank Accession number: OR047611), which indicates possible dog maternity.
Haplotype analysis revealed that out of the 31 animals in this study, 30 belong to the same haplogroup, with the exception of the animal with dog mitochondria.(Supplementary Table 1).This result confirms the previous findings that golden jackals show a high degree of genetic uniformity 52 .

Microsatellite analysis, structure and kinship results
All loci were polymorphic, with the mean number of alleles reaching 5.52, with average expected-and observed heterozygosity being 0.682 and 0.660, respectively (Supplementary Tables 2, 3, 4), all loci were therefore included in subsequent multi-locus analyses.Using 20 polymorphic microsatellite markers, we determined the genetic distances of our samples from dog and golden jackal samples.Structure results showed possible F1 hybridization in three cases (Fig. 4).www.nature.com/scientificreports/At K = 2, the reference populations separated completely, and all animals in the agouti and white group belonged to the jackal population.The two black and the mottled jackals could not be assigned to the reference groups with an assignment probability of 0.15 and 0.85.
Principal Coordinates Analyses show that the white animals are in the same genetic cluster as the agouticoloured jackals, whereas the black and mottled animals fall between the dogs and golden jackals (Fig. 5).
Using STRU CTU RE for clustering analysis, we found that none of the artificial genotypes in the F1, F2, and F3 groups could be clustered into either parental cluster.Supplementary Table 5 and Fig. 6 show the results of the artificial genotypes clustering analyses.
We performed kinship analysis with monogamous settings, to determine whether there is a relationship between the white, white carrying, and hybrid animals (n = 13).We found three family clusters between the white and white carrying animals with one likely (Probability > 0.75, 6647CaW/6648CaW/6651CaW) sibling group and two possible sibling groups (Probability > 0.5, 4744CaW/6343CaW and 4802CaA/6392CaW) (Supplementary Table 6).
One cluster (6647CaW/6648CaW/6651CaW) showed close sibling or parent-offspring relationship, which was confirmed by the place and time of the catch-all animals were the same age and killed in the same den at the same time.

Discussion
With the recent resettlement of golden jackals in the Hungarian lowland, where intensive agricultural involvement and stray or free-ranging dogs are present in areas with low population density, jackals have many opportunities for admixture with local dogs.Since hybridization has not been studied in this area, our research highlights the possibility that there might have been possible earlier hybridization events between dogs and golden jackals.
Analysing the MC1R gene of the animals in this study, we found that the white animals (n = 8) are all homozygous for the R306Ter (c.916C>T) mutation that results in an early stop codon, leading to an entirely pheomelanistic, white colouration, but they are also homozygous for the c.476C>A, a proline to glutamine change.Compared to MC1R sequences downloaded from the NCBI database, we found that the SNP has been described by Wang et al. 53 , and it is present in the Kunming Dog population, as well as in dog breeds from FCI Group 7., Ancient breeds: Alaskan Malamutes, Siberian Huskies, Shar peis, Chowchows, Akitas, Eurasiers, Tibetan mastiffs, it was also found in salukis, beagles and chihuahuas 54 , but it could not be found in other publicly available dog, wolf and coyote MC1R sequences.Since mainly ancient type dogs carry the same mutations as our white samples, it is possible that the "e" allele entered the gene pool through an earlier hybridization event with an ancient type dog.
We also found an additional SNP in the MC1R sequences: at c.475 there is a C>T, proline to serine amino acid change, which appeared only in agouti jackals, and was not found it in the dog, wolf, and coyote MC1R sequences available in the NCBI database.
Galov et al. in 34 found several wild golden jackal-dog hybrids and confirmed their hybrid status through genetic evidence, supporting the assumption that dogs and golden jackals are capable of interbreeding in areas, where their territories overlap.Stronen et al. proved 16 that human agricultural intervention can induce hybridization in otherwise sympatric species.Our findings also confirm that hybridization naturally occurs between jackals and dogs, and phenotypic changes can be expected, the most spectacular being the black coat colouration caused by a deleterious mutation in CBD103 originating from dogs, that was found by both Galov et al. and ourselves. .Principal coordinates analysis of the animals in this study.Yellow = reference dogs, pink = reference jackals, red = agouti group, green = white group, blue = black and mottled animals.The white animals are all grouped together with the reference and agouti groups, whereas the coloured animals fall between the reference dog and jackal groups.
Since the hybrid animals in our study are not relatives, we can here report three different hybridization events, which raises the question how often it happens, and what happened to the littermates of the animals, whether they were unreportedly shot or they are still in the breeding population.The case of the mottled, merle animal sheds light on the dangers of hybridization with dogs-longer SILV SINE insertions in homozygous form can cause developmental issues, like hearing loss and micropthalmia 55 , and since dogs carry at least 152 possible genetic health issues 56 , most of them recessive, developing symptoms after reaching sexual maturity, they are capable of hiding in the gene pool for generations, leading to possible regression of health in isolated populations.
Barash et al. 27 found evidence for possible hybridization of golden jackals with dogs in Israel that confirms our findings of naturally occurring dog-jackal hybrids, and found introgression with African wolves (Canis lupaster), which shows that golden jackals are prone to interbreeding with other Canid species, where their habitats overlap.In Europe, besides free roaming dogs, the only other Canid species golden jackals might share a habitat with are grey wolves (Canis lupus), but wolves possibly have a key role in the regulation of the golden jackal population 3 , and we have found no evidence of hybridization between the two species.
Coyotes' and wolves' hybridization with dogs has already raised questions regarding the behaviour of the introgressed populations-they become bolder and possibly more dependent on human resources 57 ; however, the effects of hybridization on the opportunistic lifestyle of golden jackals is still unknown and needs more investigation.
In conclusion, we identified the mutation responsible for white coat in golden jackals, which is the same as in dogs.This allele was introduced to the species possibly through an earlier hybridization event with an ancient type dog.We also identified three dog-golden jackal hybrid animals, two of them are heterozygous for the CBD103 ΔG23 mutation causing black coat, and one of them is heterozygous for the SILV SINE insertion causing merle pattern in dogs.Our findings confirm that golden jackals interbreed with dogs and hybridization with dogs results in phenotypic change, which in recessive form can stay hidden for generations.A recessive allele for a hereditary disease entering the population from the dogs' side could pose a danger for isolated jackal populations.

Sampling and genomic DNA extraction
Agouti (n = 20), white (n = 8), black (n = 2) and mottled (n = 1) golden jackal (Fig. 7) muscle and liver tissue samples were collected from legally hunted, free-ranging animals between 2014 and 2021 at hunting sites localized in the southern regions of Hungary (Somogy county, Baranya county, Csongrád-Csanád county) (Figs. 8, 9).As reference samples for studying hybridization levels, hair with follicles (n = 27) from legally shot, freeranging golden jackals from observed populations, and samples from mixed breed and pedigree dogs (n = 23) were collected and stored at − 20 °C until further laboratory procedure.
Genomic DNA isolation was performed using the MagCore HF16 Automated Nucleic Extractor® with the MagCore Genomic DNA Tissue Kit® (RBC Bioscience Corp., Taiwan), according to the manufacturer's protocol; the genomic DNA samples were stored at − 20 °C.

Genetic analysis of colour
MC1R sequencing was performed with D and E primers described by Newton et al. 38 (primer D: 5′-GGT CAT TGC TGA GCT GAC AC-3′, primer E: 5′-GAG ATG CTG TCC AGT AGT CCC-3′).The volume of the PCR mixture was 25 µl, using Multiplex MasterMix (Qiagen Gmbh, Germany); 0.4 µl of each primer diluted to 10 µM, and 120 ng of DNA diluted to 15 ng/µl.PCR was conducted in a GeneExplorer instrument (Hangzhou Bioer Technology Co., China).The PCR profile was the following: 95 °C for 15 min, 40 cycles of 94 °C for 30 s, 64 °C for 60 s, 72 °C for 2 min, followed by a final extension step of 72 °C for 5 min.The 1347-bp-long PCR products were purified using NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel Gmbh, Germany); Sanger-sequencing was executed using BigDye Terminator 3.1 Cycle Sequencing Kit and 3500xl Genetic Analyzer instrument (Applied Biosystems, USA).MC1R sequences were aligned using SeqMan Pro (DNAStar, Inc), and the alignments were checked manually.We used the MC1R sequence of ROS_Cfam_1.0 (NC_051809.1:c63923224-63922271, Labrador retriever) as a reference.Amplification of the SILV SINE insertion was done using the method and primers described by Langevin et al. 51 .We used Qiagen Multiplex MasterMix (Qiagen Gmbh, Germany) according to the producer's instructions.The length of the SINE insertion was determined using Geneious Prime® (Biomatters Ltd).

Hybridization analyses
To detect possible hybridization with dogs and conclude kinship, we performed microsatellite and mitochondrial sequence analysis on the samples to detect the direction of hybridization.
Amplification of the hypervariable mitochondrial control region (D-loop) was performed with primers described by Fabbri et al. 58 (5′-TCC CTG ACA CCC CTA CAT TC-3′, 5′-CGT TGC GGT CAT AGG TGA G-3′) resulting in a 519-basepair amplicon.The reaction mixture volume was 20 µl, it contained 3 µl of DNA diluted to the concentration of 15 ng/µl, 10 µl of Phire Animal Tissue PCR Buffer, 0.4 µl of Phire HS II DNA polymerase enzyme, 0.6 µl of each primer diluted to 10 µM and 5.4 µl nuclease-free water.PCR was conducted in a GeneExplorer instrument (Hangzhou Bioer Technology Co., China).The PCR profile was the following: 98 °C for 30 s, 35 cycles of 98 °C for 20 s, 62.5 °C for 30 s, 72 °C for 70 s, and final extension at 72 °C for 5 min.PCR products were purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel Gmbh, Germany); Sanger-sequencing was executed using BigDye Terminator 3.1 Cycle Sequencing Kit and 3500xl Genetic Analyzer instrument (Applied Biosystems, USA).
The sex of the animals was identified by hunters and confirmed with the amelogenin gene 62 .The reaction mixture volume was 25 µl, containing Multiplex MasterMix (Qiagen Gmbh, Germany), 120 ng of DNA, optimalized amounts of primers at 10 µM concentration, and nuclease-free water.PCR was conducted in a GeneExplorer instrument (Hangzhou Bioer Technology Co., China).The PCR profile for all multiplexes was the following: 96 °C for 15 min; 40 cycles of 95 °C for 30 s, 58 °C for 60 s, 72 °C for 60 s, and final extension

Figure 4 .
Figure 4. Result of the STRU CTU RE analyses at K = 2. Group 1 = reference dogs (n = 23), Group 2 = reference jackals (n = 27), Group 3 = agouti jackals (n = 20), Group 4 = white jackals (n = 8), Group 5 = Black and mottled animals (n = 3).The Delta K value shows the most likely number of clusters, which is 2 in this case.The colour red indicates clustering with the dog group; green is the colour of the golden jackal cluster.In group 5, the individuals show approximately 50% clustering to both groups, so they cannot be clustered into either.

Figure 5
Figure5.Principal coordinates analysis of the animals in this study.Yellow = reference dogs, pink = reference jackals, red = agouti group, green = white group, blue = black and mottled animals.The white animals are all grouped together with the reference and agouti groups, whereas the coloured animals fall between the reference dog and jackal groups.

Figure 9 .
Figure 9. Sample collection sites of the coloured animals.CRO Croatia, SRB Serbia, RO Romania.